DEVELOPMENT OF EXPRESSION CLONING SYSTEM FOR ONCOGENE CDNAS
癌基因 CDNAS 表达克隆系统的开发
基本信息
- 批准号:3916899
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:
项目摘要
We are developing a cDNA expression cloning system for isolation
of dominant as well as recessive oncogenes. ln this system. cDNA
libraries are constructed in eukaryotic expression vectors using
poly(A)-selected RNAs from transformants or tumors. The library
DNA can then be used to transfect NIH/3T3 cells. cDNA clones will
be recovered from resulting foci, and their structures analyzed.
In order to make expression cloning feasible for this purpose, cDNA
libraries containing complete coding sequences will be necessary.
At this point, we have developed a high efficiency cDNA cloning
system which can direct the orientation of inserts in eukaryotic
expression vectors which possess large cloning capacities. Our
lambda-plasmid composite vectors contain a retroviral long terminal
repeat (LTR) promoter to express cDNA and a simian virus 40 (SV40)
early promoter-driven neo gene as a eukaryotic cell selection
marker. cDNA was synthesized from a linker-primer containing the
site for SfiI, an infrequent cutter of DNA. An adaptor was ligated
at both ends of the double-stranded cDNA molecules, cleaved by
SfiI, and ligated with the lambda vector arms prepared by cutting
at two different SfiI sites. Due to the directional cloning
strategies and non-symmetrical structure of sticky ends of both the
vector and insert DNAs, the efficiencies of 10 to 100 million
plaque-forming units of phages were obtained from one microgram of
poly(A)-selected RNA. Furthermore, we have shown that our
libraries contain cDNAs for several growth factors and receptors
that are nearly full length molecules of 2.5 to 6.5 kb, at high
frequencies.
我们正在开发一个cDNA表达克隆系统,
显性和隐性致癌基因的结合。 在这个系统里。cDNA
文库在真核表达载体中构建,
来自转化体或肿瘤的poly(A)选择的RNA。 图书馆
然后可以使用DNA转染NIH/3 T3细胞。 cDNA克隆将
从所得病灶中回收,并分析其结构。
为了使为此目的的表达克隆变得可行,cDNA
将需要含有完整编码序列的文库。
至此,我们建立了一个高效的cDNA克隆
在真核生物中可以指导插入物方向的系统
具有大克隆能力的表达载体。 我们
双链-质粒复合载体含有逆转录病毒长末端
重复序列(LTR)启动子表达cDNA和猿猴病毒40(SV 40)
早期启动子驱动的neo基因作为真核细胞选择
标记。 cDNA由含有以下的接头引物合成:
SfiI位点,一种罕见的DNA切割器。 连接接头
在双链cDNA分子的两端,
SfiI,并与通过切割制备的λ载体臂连接
在两个不同的SfiI站点。 由于定向克隆
策略和粘性末端的非对称结构,
载体和插入DNA,效率为10至100万
从1微克的
poly(A)-选择性RNA。 此外,我们还表明,
文库含有几种生长因子和受体的cDNA
它们几乎是2.5到6.5 kb的全长分子,
频率.
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('T MIKI', 18)}}的其他基金
SIGNAL TRANSDUCTION THROUGH THE ECT2 ONCOGENE PRODUCT
通过 ECT2 癌基因产品的信号转导
- 批准号:
3774842 - 财政年份:
- 资助金额:
-- - 项目类别:
ISOLATION OF NOVEL ONCOGENES BY AN EFFICIENT EXPRESSION CLONING SYSTEM
通过高效表达克隆系统分离新型癌基因
- 批准号:
3752681 - 财政年份:
- 资助金额:
-- - 项目类别:
DEVELOPMENT OF EXPRESSION CLONING SYSTEM FOR ONCOGENE CDNAS
癌基因 CDNAS 表达克隆系统的开发
- 批准号:
3838399 - 财政年份:
- 资助金额:
-- - 项目类别:
CLONING AND CHARACTERIZATION OF NEW PROTEIN TYROSINE PHOSPHATASES
新蛋白酪氨酸磷酸酶的克隆和表征
- 批准号:
3774919 - 财政年份:
- 资助金额:
-- - 项目类别:
CHARACTERIZATION OF TST, THE EIGHTH MEMBER OF THE FGF GENE FAMILY
FGF 基因家族第八个成员 TST 的特征
- 批准号:
3752787 - 财政年份:
- 资助金额:
-- - 项目类别:
DEVELOPMENT OF EXPRESSION CLONING SYSTEM FOR ONCOGENE CDNAS
癌基因 CDNAS 表达克隆系统的开发
- 批准号:
3874710 - 财政年份:
- 资助金额:
-- - 项目类别:
DEVELOPMENT OF EXPRESSION CLONING SYSTEM FOR ONCOGENE CDNAS
癌基因 CDNAS 表达克隆系统的开发
- 批准号:
3853489 - 财政年份:
- 资助金额:
-- - 项目类别:
ISOLATION OF NOVEL ONCOGENES BY AN EFFICIENT EXPRESSION CLONING SYSTEM
通过高效表达克隆系统分离新型癌基因
- 批准号:
5201509 - 财政年份:
- 资助金额:
-- - 项目类别:
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